Screening methods for cognitive enhancers

ABSTRACT

The present invention provides methods for identifying cognitive enhancers able to enhance CREB pathway function. Cognitive enhancers identified in accordance with the invention can be used in rehabilitating an animal with cognitive dysfunctions and for enhancing memory or normal cognitive performance (ability or function) in a normal animal.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/404,620, filed Aug. 19, 2002 and U.S. Provisional Application No.60/406,405, filed Aug. 26, 2002. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

An estimated 4 to 5 million Americans (about 2% of all ages and 15% ofthose older than age 65) have some form and degree of cognitive failure.Cognitive failure (dysfunction or loss of cognitive functions, theprocess by which knowledge is acquired, retained and used) commonlyoccurs in association with central nervous system (CNS) disorders orconditions, including age-associated memory impairment, delirium(sometimes called acute confusional state), dementia (sometimesclassified as Alzheimer's or non-Alzheimer's type), Alzheimer's disease,Parkinson's disease, Huntington's disease (chorea), cerebrovasculardisease (e.g., stroke, ischemia), affective disorders (e.g.,depression), psychotic disorders (e.g., schizophrenia, autism (Kanner'sSyndrome)), neurotic disorders (e.g., anxiety, obsessive-compulsivedisorder), attention deficit disorder (ADD), subdural hematoma,normal-pressure hydrocephalus, brain tumor, head or brain trauma.

Cognitive dysfunction is typically manifested by one or more cognitivedeficits, which include memory impairment (impaired ability to learn newinformation or to recall previously learned information), aphasia(language/speech disturbance), apraxia (impaired ability to carry outmotor activities despite intact motor function), agnosia (failure torecognize or identify objects despite intact sensory function),disturbance in executive functioning (i.e., planning, organizing,sequencing, abstracting).

Cognitive dysfunction causes significant impairment of social and/oroccupational functioning, which can interfere with the ability of anindividual to perform activities of daily living and greatly impact theautonomy and quality of life of the individual. Thus, there isconsiderable interest in identifying clinical candidates for use inrehabilitating an animal with any form of cognitive dysfunction.

SUMMARY OF THE INVENTION

The present invention relates to high throughput cell-based methods(assays) to identify or screen for cognitive enhancers that act byincreasing CREB pathway function. The invention enables theidentification of cognitive enhancers that have little to no effect onCREB pathway function alone, but act to increase (enhance) CREB pathwayfunction in combination with a CREB function stimulating agent.Cognitive enhancers identified in accordance with the invention areexpected to yield effective clinical candidates for use inrehabilitating an animal with cognitive dysfunctions and for use inenhancing memory or normal cognitive performance (ability or function)in a normal animal. “Cognitive enhancers” are also referred to herein as“compounds able to enhance CREB pathway function” and “CREB pathwayenhancing drugs”. “CREB function stimulating agents” are also referredto herein as “agents that stimulate CREB pathway function”. It isunderstood that, in certain instances, a cognitive enhancer identifiedin accordance with the present invention can be a CREB functionstimulating agent. Thus, a CREB function stimulating agent may beidentified as a cognitive enhancer using the methods described herein.

As described herein, methods for identifying or screening for cognitiveenhancers comprise a primary screen, a secondary screen and a tertiaryscreen. Preferably, the primary screen is a cell-based method used toidentify candidate compounds; the secondary screen is a cell-basedmethod used to identify confirmed candidate compounds; and the tertiaryscreen uses a behavior model to identify cognitive enhancers.

The primary screen comprises: (a) contacting host cells (particularlycells of neural origin (e.g., neuroblastomas, neural stem cells))comprising an indicator gene operably linked to a CRE promoter with atest compound and with a suboptimal dose of a CREB function stimulatingagent (e.g., forskolin); (b) determining indicator activity in hostcells which have been contacted with the test compound and with the CREBfunction stimulating agent, (c) comparing the indicator activitydetermined in step (b) with the indicator activity in control cellswhich have been contacted with the CREB function stimulating agent andwhich have not been contacted with the test compound (i.e., controlcells which have been contacted with the CREB function stimulating agentalone); (d) selecting the test compound if (1) the indicator activitydetermined in step (b) is statistically significantly increased relativeto the indicator activity in the control cells of step (c); and (2) theindicator activity in control cells which have not been contacted withthe CREB function stimulating agent and which have been contacted withthe test compound (i.e., control cells which have been contacted withtest compound alone) is not statistically significantly differentrelative to the indicator activity in control cells which have not beencontacted with either the CREB function stimulating agent or the testcompound (i.e., control cells which have been contacted with nothing);(e) repeating steps (a) to (d) with a range of different concentrations(e.g., 2 or more) of the test compound selected in step (d); and (f)selecting the test compound if: (1) the indicator activity isproportionally statistically significantly increased in the range ofdifferent concentrations for said test compound relative to theindicator activity in the control cells which have been contacted withthe CREB function stimulating agent alone; and (2) the indicatoractivity in control cells to which have been introduced the range ofdifferent concentrations of the test compound alone is not significantlydifferent relative to the indicator activity in control cells which havenot been contacted with either the CREB function stimulating agent orthe test compound, wherein the test compound is identified as acandidate compound. In a particular embodiment, host cells are contactedwith the test compound prior to contact with the CREB functionstimulating agent. In another embodiment, the indicator gene encodesluciferase. An indicator gene operably linked to a CRE promoter is alsoreferred to herein as a CRE-mediated indicator gene. A CRE-mediatedindicator gene is an example of a CRE-mediated transgene.

Alternatively, the primary screen comprises: (a) contacting host cells(particularly cells of neural origin (e.g., neuroblastomas, neural stemcells)) with a test compound and with a suboptimal dose of a CREBfunction stimulating agent (e.g., forskolin); (b) assessing endogenousCREB-dependent gene expression in the host cells which have beencontacted with the test compound and with the CREB function stimulatingagent; (c) comparing endogenous CREB-dependent gene expression assessedin step (b) with endogenous CREB-dependent gene expression in controlcells which have been contacted with the CREB function stimulating agentand which have not been contacted with the test compound (i.e., controlcells which have been contacted with the CREB function stimulating agentalone); (d) selecting the test compound if (1) the endogenousCREB-dependent gene expression determined in step (b) is statisticallysignificantly increased relative to the endogenous CREB-dependent geneexpression in the control cells of step (c); and (2) the CREB-dependentgene expression in control cells to which have not been contacted withthe CREB function stimulating agent and which have been contacted withthe test compound (i.e., control cells which have been contacted withtest compound alone) is not statistically significantly differentrelative to the CREB-dependent gene expression in control cells whichhave not been contacted with either the CREB function stimulating agentor the test compound (i.e., control cells which have been contacted withnothing); (e) repeating steps (a) to (d) with a range of differentconcentrations (e.g., 2 or more) of the test compound selected in step(d); and (f selecting the test compound if: (1) the CREB-dependent geneexpression is proportionally statistically significantly increased inthe range of different concentrations for said test compound relative tothe CREB-dependent gene expression in the control cells which have beencontacted with the CREB function stimulating agent alone; and (2) theCREB-dependent gene expression in control cells to which have beenintroduced the range of different concentrations of the test compoundalone is not significantly different relative to the CREB-dependent geneexpression in control cells which have not been contacted with eitherthe CREB function stimulating agent or the test compound, wherein thetest compound is identified as a candidate compound. In a particularembodiment, host cells are contacted with the test compound prior tocontact with the CREB function stimulating agent.

The secondary screen comprises: (a) contacting cells of neural origin(particularly primary neurons (e.g., primary hippocampal cells)) with acandidate compound identified in the primary screen and with asuboptimal dose of a CREB function stimulating agent; (b) assessingendogenous CREB-dependent gene expression in the cells which have beencontacted with the candidate compound and with the CREB functionstimulating agent; and (c) comparing endogenous CREB-dependent geneexpression assessed in step (b) with endogenous CREB-dependent geneexpression in control cells which have been contacted with the CREBfunction stimulating agent and which have not been contacted with thecandidate compound (i.e., control cells which have been contacted withCREB function stimulating agent alone). A statistically significantdifference in CREB-dependent gene expression assessed in step (b)compared to the CREB-dependent gene expression in control cells whichhave been contacted with CREB function stimulating agent alone and nosignificant difference in CREB-dependent gene expression in controlcells which have not been contacted with the CREB function stimulatingagent and which have been contacted with the candidate compound (i.e.,control cells contacted with candidate compound alone) relative toCREB-dependent gene expression in control cells which have not beencontacted with either the CREB function stimulating agent or thecandidate compound (i.e., control cells contacted with nothing)identifies the candidate compound as a confirmed candidate compound. Ina particular embodiment, cells are contacted with the candidate compoundprior to contact with the CREB function stimulating agent.

Alternatively, the secondary screen comprises: (a) contacting cells ofneural origin particularly primary neurons (e.g., primary hippocampalcells)) comprising an indicator gene operably linked to a CRE promoterwith a candidate compound identified in the primary screen and with asuboptimal dose of a CREB function stimulating agent; (b) determiningindicator activity in the cells which have been contacted with thecandidate compound and with the CREB function stimulating agent; and (c)comparing indicator activity assessed in step (b) with indicatoractivity in control cells which have been contacted with the CREBfunction stimulating agent and which have not been contacted with thecandidate compound (i.e., control cells which have been contacted withCREB function stimulating agent alone). A statistically significantdifference in indicator activity determined in step (b) compared to theindicator activity in control cells which have been contacted with CREBfunction stimulating agent alone and no significant difference inindicator activity in control cells which have been contacted with theCREB function stimulating agent and which have not been contacted withthe candidate compound (i.e., control cells contacted with candidatecompound alone) relative to indicator activity in control cells whichhave not been contacted with either the CREB function stimulating agentor the candidate compound (i.e., control cells contacted with nothing)identifies the candidate compound as a confirmed candidate compound. Ina particular embodiment, cells are contacted with the candidate compoundprior to contact with the CREB function stimulating agent.

In a particular embodiment, the cells of neural origin used in thesecondary screen are different from the host cells used in the primaryscreen. In another embodiment, the endogenous CRE-mediated gene or theCRE-mediated transgene in the secondary screen is different from theCRE-mediated transgene or the endogenous CRE-mediated gene in theprimary screen. For example, in one embodiment the host cells in theprimary screen are neuroblastomas and the cells in the secondary screenare not neuroblastomas. In another embodiment, the CRE-mediatedtransgene in the primary screen is luciferase (CRE operably linked tothe luciferase gene) and the CRE-mediated transgene in the secondaryscreen is not luciferase. In a particular embodiment, the host cells inthe primary screen are proliferating cells (such as neuroblastomas andneural stem cells) and the cells in the secondary screen arenonproliferating, differentiated cells of neural origin (such as neuronsor glial cells). In another particular embodiment, the CRE-mediated genein the primary screen is a CRE-mediated indicator gene (a CRE-mediatedtransgene) and the CRE-mediated gene in the secondary screen is anendogenous CRE-mediated gene.

In one embodiment, the tertiary screen is a behavioral method forassessing long term memory formation in an animal comprising: (a)administering an effective amount of a confirmed candidate compoundidentified in the secondary screen to the animal (e.g., human, othermammal, vertebrate or invertebrate); (b) training the animaladministered the confirmed candidate compound under conditionsappropriate to produce long term memory formation in the animal; (c)assessing long term memory formation in the animal trained in step (b);and (d) comparing long term memory formation assessed in step (c) withlong term memory formation produced in the control animal to which theconfirmed candidate compound has not been administered If an enhancementis noted in long term memory formation assessed in the animal treatedwith the confirmed candidate compound relative to the long term memoryformation assessed in the control animal, the confirmed candidatecompound is identified as a cognitive enhancer. Tertiary screens withvery similar protocols are available using behavioral methods (models)for other cognitive dysfunctions.

The invention also relates to methods for assessing the effect of acompound on CREB-dependent gene expression (CRE-mediated geneexpression) comprising: (a) contacting cells of neural originparticularly primary neurons (e.g., primary hippocampal cells) with acompound to be assessed and with a suboptimal dose of a CREB functionstimulating agent; (b) assessing endogenous CREB-dependent geneexpression in the cells which have been contacted with the compound andwith the CREB function stimulating agent; and (c) comparing endogenousCREB-dependent gene expression assessed in step (b) with endogenousCREB-dependent gene expression in control cells which have beencontacted with the CREB function stimulating agent and which have notbeen contacted with the compound (i.e., control cells which have beencontacted with CREB function stimulating agent alone). A statisticallysignificant difference in CREB-dependent gene expression assessed instep (b) compared to the CREB-dependent gene expression in control cellswhich have been contacted with CREB function stimulating agent alone andno significant difference in CREB-dependent gene expression in controlcells which have not been contacted with the CREB function stimulatingagent and which have been contacted with the compound (i.e., controlcells contacted with compound alone) relative to CREB-dependent geneexpression in control cells which have not been contacted with eitherCREB function stimulating agent or the compound (i.e., control cellscontacted with nothing) identifies the compound as one having an effecton CREB-dependent gene expression. In a particular embodiment, cells arecontacted with the compound to be assessed prior to contact with theCREB function stimulating agent. Preferably, the compound to be assessedis a candidate compound identified in the primary screen.

Alternatively, methods for assessing the effect of a compound onCREB-dependent gene expression comprise: (a) contacting cells of neuralorigin (particularly primary neurons (e.g., primary hippocampal cells)comprising an indicator gene operably linked to a CRE promoter with acompound to be assessed and with a suboptimal dose of a CREB functionstimulating agent; (b) determining indicator activity in the cells whichhave been contacted with the compound and with the CREB functionstimulating agent; and (c) comparing indicator activity determined instep (b) with indicator activity in control cells which have beencontacted with the CREB function stimulating agent and which have notbeen contacted with the compound (i.e., control cells which have beencontacted with CREB function stimulating agent alone). A statisticallysignificant difference in indicator activity determined in step (b)compared to the indicator activity in control cells which have beencontacted with CREB function stimulating agent alone and no significantdifference in indicator activity in control cells which have not beencontacted with the CREB function stimulating agent and which have beencontacted with the compound (i.e., control cells contacted with compoundalone) relative to indicator activity in control cells which have notbeen contacted with either CREB function stimulating agent or thecompound (i.e., control cells contacted with nothing) identifies thecompound as one having an effect on CREB-dependent gene expression. In aparticular embodiment, cells are contacted with the compound to beassessed prior to contact with the CREB function stimulating agent.Preferably, the compound to be assessed is a candidate compoundidentified in the primary screen.

In a particular embodiment, the cells used in the methods for assessingthe effect of a compound on CREB-dependent gene expression are differentfeom the host cells in the primary screen. In another embodiment, theendogenous CRE-mediated gene or the CRE-mediated transgene used in themethods for assessing the effect of a compound on CREB-dependent geneexpression are different from the CRE-mediated transgene or theendogenous CRE-mediated gene in the primary screen.

The invention further relates to methods for assessing the effect of acognitive enhancer on long term memory formation in an animalcomprising: (a) administering an effective amount of cognitive enhancerto an animal (e.g., human, other mammal, vertebrate or invertebrate);(b) training the animal administered the cognitive enhancer underconditions appropriate to produce long term memory formation in theanimal; (c) assessing long term memory formation in the animal trainedin step Ado), and (d) comparing long term memory formation assessed instep (c) with long term memory formation produced in the control animalto which the cognitive enhancer has not been administered. If adifference is noted in long term memory formation assessed in the animaltreated with the cognitive enhancer relative to the long term memoryformation assessed in the control animal, the cognitive enhancer can becategorized as enhancing (improving, increasing) long term memoryformation in the animal.

Cognitive enhancers identified in accordance with the invention can beused to reduce the duration and/or number of training sessions requiredfor the induction in a specific neuronal circuit(s) of a pattern ofneuronal activity or to reduce the duration and/or number of trainingsessions required to induce CREB-dependent long term structural/function(i.e., long-lasting) change among synaptic connections of the neuronalcircuit. Cognitive enhancers identified in accordance with the inventioncan be used to reduce the number of training sessions required to inducea performance gain relative to that obtained with training alone or toenable shorter or no rest intervals between training sessions to inducea performance gain or to increase the overall level of performance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the results of drug-induced (partial)enhancement of memory in normal mice by rolipram, a prototypicalphosphodiesterase (PDE) inhibitor. Five-day retention after weaktraining (2×) is lower than strong training (5×). Only memory after weaktraining is enhanced by bilateral hippocampal injections (1 μl) ofrolipram immediately after training. Rolipram reduced the number oftraining sessions required to produce memory. %/Freezing=% Observationtime mice spent frozen/motionless.

FIG. 2 is a schematic diagram showing that experience-dependent changesin neural activity modulate gene expression. Some of these changes ingene expression yield structural and functional changes in the synapticconnections among neurons. In this manner, neural circuitry isconstantly fine-tuned to optimize perception cognition and behavioralresponses.

FIG. 3 is a bar graph showing the results of a CREB Locked Nucleic Acid(LNA) antisense screen. The CREB LNB oligos are numbered in the orderthat they were designed. CREB LNB oligos that were not screened (i.e.,CREB 4, 10, 11, 13 and 14) are not shown.

DETAILED DESCRIPTION OF THE INVENTION

The CREB transcription factor plays a primary role in the formation oflong-term memory and the underlying long-term synaptic plasticity.Behavior-genetic studies of Pavlovian olfactory learning in Drosophilahave established CREB to be a central switch for the conversion of newlyacquired information from short-term memory to long-term memory (Tully,T. et al., Proc. Natl. Acad. Sci. USA, 94(9):4239-4241 (1997)). Spacedtraining after a Pavlovian odor-shock association task induces a proteinsynthesis-dependent long-term memory (Tully, T. et al., Cell,79(1):35-47 (1994)), the formation of which is specifically blocked byinduced expression of a CREB repressor transgene (Yin, J. C. et al.,Cell, 79(1):49-58 (1994)). Learning and short-term (earlier) memory arenormal in these transgenic flies (Tully, T. et al., Cell, 79(1):35-47(1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)). In contrast tothese loss-of-function results, induced expression of a CREB activatortransgene enhances long-term memory specifically by abrogating therequirements for repetitive training sessions and for a rest intervalbetween each session (Yin, J. C. et al., Cell, 81(1):107-115 (1995)).One training session is sufficient to form maximal long-term memory intransgenic flies overexpressing CREB activator (Yin, J. C. et al., Cell,81(1):107-115 (1995)). More long-term memory is not produced. Rather,long-term memory is induced with less practice.

This memory enhancement also is specific to the temporal association oftwo stimuli. High levels of CREB activator were induced in all cells ofthe fly due to the use of a heat-shock promoter with the transgene. Inthe absence of training, no measurable effect on the flies' behavior wasobserved. Moreover, spaced presentations of odor and shock, unpaired intime, failed to produce long-term memory in CREB-activator flies (fliesinduced to express high levels of CREB activator). These results suggestthat associative learning induces the upstream signaling required toactivate (phosphorylate) the CREB switch during olfactory long-termmemory formation.

A growing body of evidence extends these results from invertebrates tomammals. For example, in Aplysia, molecular manipulations of CREBexpression, similar to those in flies, suppress or enhance (1) long-termmemory of a facilitatory electrophysiological response at a sensorimotormonosynapse in cell culture and (2) the synaptic connections betweensensory and motor neurons that are normally produced after spacedapplications of the facilitatory stimulus (Bartsch, D. et al., Cell,83(6):979-992 (1995)). In a study focused on a form of traumatic memory,referred to as fear conditioning (Bourtchuladze, R P et al., Cell,79(1):59-68 (1994)), mutant mice carrying a partial knock-out of CREBshowed normal learning and short-term memory but long-term memory wasabolished. In contrast, normal mice required only one training trial toform a long-term memory of this experience. These results suggest that(1) for the fear conditioning experiment, normal mice formed long-termmemory more like the CREB-activator flies and (2) the CREB mutation inmice was partial, reducing the amount of activator isoform(s) relativeto repressor isoform(s). This indicated that CREB mutant mice may have afunctional CREB switch that is set to a level similar to that in normalflies. As such, spaced training would rescue the long-term memorydeficit in CREB mutant mice. In fact, Kogan et al. showed that long-termmemory can form normally in CREB mutant mice subjected to spaced, butnot massed, training, thereby providing strong support for the CREBswitch model (Kogan, J. H. et al., Curr. Biol., 7(1):1-1 (1997)).

In rats, injections of antisense RNA oligonucleotides into hippocampusor amygdala block long-term memory formation of two different tasks thatare dependent on activity in these anatomical regions, respectively(Guzowski, J. F. and McGaugh, J. L., Proc. Natl. Acad. Sci. USA,94(6):2693-2698 (1997); and Lamprecht, R. et al., J. Neurosci.,17(21):8443-8450 (1997)). In particular, the RNA antisense experimentsrevealed a role for CREB during long-term memory formation of awater-maze task (hippocampal-dependent) and conditioned taste aversion(amygdala-dependent) in rats (Guzowski, J. F. and McGaugh, J. L., Proc.Natl. Acad. Sci. USA, 94(6):2693-2696 (1997); and Lamprecht, R. et al.,J. Neurosci., 17(21):8443-8450 (1997)).

More recent experiments have over expressed CREB activator in theamygdala and observed enhanced memory formation of a fear-potentiatedstartle response in rats, with a CREB signature similar to that observedin flies (Josselyn, S. A. et al., Society for Neuroscience, 28th AnnualMeeting, Vol. 24, Abstract 365.10 (1998); and Josselyn, S. A. et al., J.Neurosci., 21(7):2404-2412 (2001)).

Cellular observations in mice and rats have reinforced these behavioralresults by revealing that neuronal CREB function (phosphorylation orgene-regulation) is modulated in an experience-dependent fashion (Impey,S. et al., Neuron, 16(5):973-982 (1996); Taubenfeld S. M. et al., Nat.Neurosci., 2(4):309-310 (1999); and Taubenfeld, S. M. et al., J.Neurosci., 21(1):84-91 (2001)).

In addition to being involved in the formation of experiential memories,CREB also appears to function in the developmental and cellularplasticity of several cortical regions (Ahn, S. et al., Neuron,23(3):559-568 (1999); Barth, A. L. et al., J. Neurosci.,20(11):4206-4216 (2000); Glazewski, S. et al., Cerebral Cortex,9(3):249-256 (1999); Pham, T. A. et al., Neuron, 22(1):63-72 (1999); andPham, T. A. et al., Neuron, 31(3):409-420 (2001)). Neuronal activityincreases CREB activity in cortex (Moore, A. N. et al., J. Biol. Chem.,271(24):14214-14220 (1996)). CREB also mediates developmental plasticityin hippocampus (Murphy, D. D. and Segal, M., Proc. Natl. Acad. Sci. USA,94(4):1482-1487 (1997)), in somatosensory cortex (Glazewski, S. et al.,Cerebral Cortex, 9(3):249-256 (1999)), in striatum (Liu, F. C. andGraybiel, A. M., Neuron, 17(6):1133-1144 (1996)) and in visual cortex(Pham, T. A. et al, Neuron, 22(1):63-72 (1999)). These data support theidea that CREB is a molecular switch generally involved in convertingneural activity to structural changes at synapses. As such, the CREBpathway represents (1) a significant target for new drug discovery and(2) a genetic beachhead for the identification of downstream genes thatalso participate in activity-dependent synaptic plasticity.

The present invention provides methods to identify or screen for thesedrugs, also referred to herein as cognitive enhancers. The inventionprovides high throughput cell-based methods (assays) to identify orscreen for cognitive enhancers that act by increasing CREB pathwayfunction.

Cognitive enhancers can increase, enhance or improve CREB pathwayfunction by a variety of mechanisms. For example, a cognitive enhancercan affect a signal transduction pathway which leads to induction ofCREB-dependent gene expression. Induction of CREB-dependent geneexpression can be achieved, for example, via up-regulation of positiveeffectors of CREB function and/or down-regulation of negative effectorsof CREB function. Positive effectors of CREB function include adenylatecyclases and CREB activators. Negative effectors of CREB functioninclude cAMP phosphodiesterase (cAMP PDE) and CREB repressors.

A cognitive enhancer can increase, enhance or improve CREB pathwayfunction by acting biochemically upstream of or directly acting on anactivator or repressor form of a CREB protein and/or on a CREB proteincontaining transcription complex. For example, CREB pathway function canbe affected by increasing CREB protein levels transcriptionally,post-transcriptionally, or both transcriptionally andpost-transcriptionally, by altering the affinity of CREB protein toother necessary components of the of the transcription complex, such as,for example, to CREB-binding protein (CBP protein); by altering theaffinity of a CREB protein containing transcription complex for DNA CREBresponsive elements in the promoter region; or by inducing eitherpassive or active immunity to CREB protein isoforms. The particularmechanism by which a cognitive enhancer increases, enhances or improvesCREB pathway function is not critical to the practice of the invention.

By “increase CREB pathway function” or “enhance CREB pathway function”is meant the ability to increase, enhance or improve CREB-dependent geneexpression. By “modulate CREB pathway function” is meant the ability tomodulate CREB-dependent gene expression. CREB-dependent gene expressioncan be increased, enhanced or improved by increasing endogenous CREBproduction, for example by directly or indirectly stimulating theendogenous gene to produce increased amounts of CREB, or by increasingfunctional (biologically active) CREB. See, e.g., U.S. Pat. No.5,929,223; U.S. Pat. No. 6,051,559; and International Publication No.WO96/11270 (published Apr. 18, 1996), which references are incorporatedherein in their entirety by reference. By “increasing functional(biologically active) CREB” is meant to include the ability to increaseDNA binding ability, phosphorylation state, protein stability,subcellular localization, etc. CREB-dependent gene expression can bemodulated by increasing or decreasing endogenous CREB production, forexample by directly or indirectly stimulating the endogenous gene toproduce increased or decreased amounts of CREB, or by increasing ordecreasing functional (biologically active) CREB.

As described herein, methods for identifyng or screening for cognitiveenhancers comprise a primary screen, a secondary screen and a tertiaryscreen. Preferably, the primary screen is a cell-based method used toidentify candidate compounds; the secondary screen is a cell-basedmethod used to identify confirmed candidate compounds; and the tertiaryscreen uses a behavior model to identify cognitive enhancers. Primaryand secondary screens include high throughput cell-based methods.

The primary screen comprises: (a) contacting host cells comprising anindicator gene operably linked to a CRE promoter with a test compoundand with a suboptimal dose of a stimulating agent that activatessignaling pathways onto CREB; (b) determining indicator activity in hostcells which have been contacted with the test compound and with thestimulating agent; (c) comparing the indicator activity determined instep (b) with the indicator activity in control cells which have beencontacted with the stimulating agent and which have not been contactedwith the test compound (control cells which have been contacted withstimulating agent alone); (d) selecting the test compound if (1) theindicator activity determined in step (b) is statistically significantlyincreased relative to the indicator activity in the control cells ofstep (c); and (2) the indicator activity in control cells which have notbeen contacted with the stimulating agent and which have been contactedwith the test compound (control cells contacted with test compoundalone) is not statistically significantly different relative to theindicator activity in control cells which have been contacted withneither the stimulating agent or the test compound (controls cells whichhave been contacted with nothing); (e) repeating steps (a) to (d) with arange of different concentrations of the test compound selected in step(d); and (f) selecting the test compound if: (1) the indicator activityis proportionally statistically significantly increased in the range ofdifferent concentrations of said test compound relative to the indicatoractivity in the control cells to which have been contacted with thestimulating agent alone; and (2) the indicator activity in control cellsto which have been introduced the range of different concentrations ofthe test compound alone is not significantly different relative to theindicator activity in control cells which have not been contacted witheither the stimulating agent or the test compound, wherein the testcompound is identified as a candidate compound. In a particularembodiment, the test compound is selected in step (f) if (1) theindicator activity is proportionally significantly increased in thelinear range of different concentrations for the test compound; and (2)the indicator activity in control cells to which have been introducedthe range of different concentrations of the test compound alone is notsignificantly different relative to the indicator activity in controlcells which have not been contacted with either the stimulating agent orthe test compound. In another embodiment, host cells are contacted withthe test compound prior to contact with the stimulating agent.

Alternatively, the primary screen comprises: (a) contacting host cellswith a test compound and with a suboptimal dose of a stimulating agentthat activates signaling pathways onto CREB; (b) assessing endogenousCREB-dependent gene expression in the host cells which have beencontacted with the test compound and with the stimulating agent; (c)comparing endogenous CREB-dependent gene expression assessed in step (b)with endogenous CREB-dependent gene expression in control cells whichhave been contacted with the stimulating agent and which have not beencontacted with the test compound (control cells which have beencontacted with stimulating agent alone); (d) selecting the test compoundif (1) the endogenous CREB-dependent gene expression determined in step(b) is statistically significantly increased relative to the endogenousCREB-dependent gene expression in the control cells of step (c); and (2)the CREB-dependent gene expression in control cells which have not beencontacted with the stimulating agent and which have been contacted withthe test compound (control cells which have been contacted with testcompound alone) is not statistically significantly different relative tothe CREB-dependent gene expression in control cells which have beencontacted with neither the stimulating agent or the test compound(controls cells which have been contacted with nothing); (e) repeatingsteps (a) to (d) with a range of different concentrations of the testcompound selected in step (d); and (f) selecting the test compound if:(1) the CREB-dependent gene expression is proportionally statisticallysignificantly increased in the range of different concentrations forsaid test compound relative to the CREB-dependent gene expression in thecontrol cells which have been contacted with the stimulating agentalone; and (2) the CREB-dependent gene expression in control cells towhich have been introduced the range of different concentrations of thetest compound alone is not significantly different relative to theCREB-dependent gene expression in control cells which have beencontacted with neither the stimulating agent or the test compound,wherein the test compound is identified as a candidate compound. In aparticular embodiment, the test compound is selected in step (f) if (1)the CREB-dependent gene expression is proportionally significantlyincreased in the linear range of the different concentrations for thetest compound; and (2) the CREB-dependent gene expression in controlcells to which have been introduced the range of differentconcentrations of the test compound alone is not significantly differentrelative to the CREB-dependent gene expression in control cells whichhave not been contacted with either the stimulating agent or the testcompound. In another embodiment, host cells are contacted with the testcompound prior to contact with the stimulating agent.

Preferably, the “stimulating agent that activates signaling pathwaysonto CREB” used in the primary screen is a CREB function stimulatingagent. A CREB function stimulating agent is an agent that is able tostimulates CREB pathway function. By “stimulate CREB pathway function”is meant the ability to stimulate CREB-dependent gene expression bystimulating endogenous CREB production, for example by directly orindirectly stimulating the endogenous gene to produce increased amountsof CREB, or by increasing functional (biologically active) CREB. See,e.g., U.S. Pat. No. 5,929,223); U.S. Pat. No. 6,051,559; andInternational Publication No. WO96/11270 (published Apr. 18, 1996),which references are incorporated herein in their entirety by reference.“CREB function stimulating agents” include drugs, chemical compounds,ionic compounds, organic compounds, organic ligands, includingcofactors, saccharides, recombinant and synthetic peptides, proteins,peptoids, nucleic acid sequences, including genes, nucleic acidproducts, and other molecules and compositions. CREB functionstimulating agents can be activators of adenylate cyclase 1 (AC1) (e.g.,forskolin); cell permeant cAWP analogs (e.g, 8-bromo cAW); agents(neurotransmittors) affecting G-protein linked receptor, such as, butnot limited to adrenergic receptors and opioid receptors and theirligands (e.g., isoproterenol, phenethylamines); modulators ofintracellular calcium concentration (e.g., potassium chloride,thapsigargin, N-methyl-D-aspartate (NMDA) receptor agonists); inhibitors(antagonists) of the phosphodiesterases responsible for cAMP breakdown(e.g., rolipram (which inhibits phosphodiesterase 4),iso-buto-metho-xanthine (IBMX) (which inhibits phosphodiesterases 1 and2)); modulators (agonists) of protein kinases and protein phosphatases,which mediate CREB protein activation and CREB-dependent geneexpression. CREB function stimulating agents can also be compounds whichare capable of enhancing CREB function in the central nervous system(CNS). Such compounds include, but are not limited to, compounds whichaffect membrane stability and fluidity and specific immunostimulation.

Signaling pathways that activate onto CREB include the mitogen-activatedprotein kinase (MAPK) signaling pathway and protein kinase A (PKA).Thus, stimulating agents that activate signaling pathways onto CREBinclude inhibitors of MAPK/Erk kinase (MEK). Other stimulating agentsthat activate signaling pathways onto CREB are known and readilyavailable to those skilled in the art.

In another embodiment, the primary screen can be replaced with ascreening method using Drosophila, wherein said screening methodcomprises: (a) administering a test compound to Drosophila having anindicator gene operably linked to a CRE promoter; (b) assessingindicator activity in the Drosophila to which have been administered thetest compound; and (c) comparing the indicator activity assessed in step(b) with the indicator activity in control Drosophila to which have notbeen administered the test compound. A statistically significantdifference in indicator activity in step (b) compared to the indicatoractivity in control Drosophila to which have not been administered thetest compound identifies the test compound as a candidate compound.

Host cells comprising an indicator gene operably linked to a CREpromoter can be manufactured by introducing into cells a DNA constructcomprising an indicator gene operably linked to a CRE promoter. DNAconstructs can be introduced into cells according to methods known inthe art (e.g., transformation, direct uptake, calcium phosphateprecipitation, electroporation, projectile bombardment, usingliposomes). Such methods are described in more detail, for example, inSambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition(New York: Cold Spring Harbor University Press) (1989); and Ausubel, etal., Current Protocols in Molecular Biology (New York: John Wiley &Sons) (1998).

Drosophila comprising an indicator gene operably linked to a CREpromoter can be produced as described by Belvin et al., Neuron,22(4):777-787 (1999).

DNA constructs comprising an indicator gene operably linked to a CREpromoter can be manufactured as described in, for example, Ausubel etal., Current Protocols In Molecular Biology (New York: John Wiley &Sons) (1998); and Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd edition (New York: Cold Spring Harbor University Press(1989).

As used herein, the term “promoter” refers to a sequence of DNA, usuallyupstream (5′) of the coding region of a structural gene, which controlsthe expression of the coding region by providing recognition and bindingsites for RNA polymerase and other factors which may be required forinitiation of transcription. CRE promoters are known in the art.

The term “indicator gene”, as used herein, refers to a nucleic acidsequence whose product can be easily assayed, for example,colorimetrically as an enzymatic reaction product, such as the geneencoding luciferase. Other examples of widely used indicator genesinclude those encoding enzymes, such as β-galactosidase, β-glucoronidaseand β-glucosidase; luminescent molecules, such as green fluorescentprotein and firefly luciferase; and auxotrophic markers such, as His3pand Ura3p. See, e.g., Ausubel et al., Current Protocols In MolecularBiology (New York: John Wiley & Sons, Inc.), Chapter 9 (1998)).

Cells (e.g., host cells, cells of neural origin, etc.) contacted with atest compound and/or CREB function stimulating agent will take up thetest compound and/or CREB function stimulating agent.

By “suboptimal dose of CREB function stimulating agent” is meant thatamount, or dose, of CREB function stimulating agent that is required tostimulate (induce) CREB pathway function to a level that is aboveendogenous (basal) levels, such that a further statistically significantincrease in CREB pathway function due to induction by a cognitiveenhancer can be measured and the measurement is not attributable tonatural cellular fluctuations or variations as a consequence of naturalcellular fluctuations. A suboptimal dose of CREB function stimulatingagent is that dose or concentration where the amount of the effect(indicator activity, CREB-dependent gene expression) is proportional tothe dose or concentration and the amount of the effect does not changewhen the dose or concentration changes. The suboptimal dose of CREBfunction stimulating agent is determined empirically and will varydepending upon a variety of factors, including the pharmacodynamiccharacteristics of the particular CREB function stimulating agent andthe particular cells to be contacted. For example, the suboptimal doseof CREB function stimulating agent can be determined by (a) contactingdifferent samples of a host cell comprising an indicator gene operablylinked to a CRE promoter with a different concentration of the CREBfunction stimulating agent; and (b) determining the range ofconcentrations of CREB function stimulating agent required to affectindicator activity from baseline to maximal response by assessingindicator activity in the samples of the host cell. The suboptimal doseof CREB function stimulating agent will be any concentration yielding(1) 50% or less maximal indicator activity and (2) an indicator activityabove natural cellular fluctuations. Determination of the suboptimaldose of CREB function stimulating agent is well within the ability ofthose skilled in the art. By “suboptimal dose of a stimulating agentthat activates signaling pathways onto CREB” is meant that amount, ordose, of stimulating agent that is required to stimulate (induce) asignaling pathways onto CREB.

By “range of different concentrations of the test compound” is meant 2or more (i.e., 2, 3, 4, 5, etc.) different concentrations of the testcompound. The range of concentrations selected generally flanks theconcentration of the test compound in step (a) of the primary screen. By“linear range of (different) concentrations” is meant the concentrationswhere effect (indicator activity, CREB-dependent gene expression) isincreasing with concentration but prior to when the effect is no longerchanging with concentration changes. Selecting concentration ranges iswell within the ability of those skilled in the art

By “functional (biologically active) CREB” is meant to include theprotein's DNA binding ability, phosphorylation state, protein stability,subcellular localization, etc.

“CREB-dependent gene expression” is also referred to herein as“CRE-mediated gene expression”. CREB-dependent gene expression can bedetermined by methods known in the art (e.g., Northern blot, Westernblot). Such methods are described in more detail, for example, inAusubel et al., Current Protocols In Molecular Biology (New York: JohnWiley & Sons) (1998); and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd edition (New York: Cold Spring Harbor UniversityPress (1989).

“Endogenous CRE-mediated genes” are also referred to herein as“endogenous CREB-dependent genes”. Such genes are known in the art andinclude, for example, c-fos, prodynorphin, tPA and brain-derivedneurotrophic factor (BDNF) (Barco, A. et al., Cell, 108(5):689-703(2002)). CRE-mediated genes can also be identified by those skilled inthe art using methods known and readily available in the art (see, e.g.,Ausubel et al., Current Protocols In Molecular Biology (New York: JohnWiley & Sons) (1998); and Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd edition (New York: Cold Spring Harbor UniversityPress (1989)).

The secondary screen comprises: (a) contacting cells of neural originwith a candidate compound identified in the primary screen and with asuboptimal dose of a stimulating agent that activates signaling pathwaysonto CREB; (b) assessing endogenous CREB-dependent gene expression inthe cells which have been contacted with the candidate compound and withthe stimulating agent; and (c) comparing endogenous CREB-dependent geneexpression assessed in step (b) with endogenous CREB-dependent geneexpression in control cells which have been contacted with thestimulating agent and which have not been contacted with the candidatecompound (control cells which have been contacted with stimulating agentalone). A statistically significant difference in CREB-dependent geneexpression assessed in step (b) compared to the CREB-dependent geneexpression in control cells identifies the candidate compound as aconfirmed candidate compound. Preferably, no significant difference isobtained in CREB-dependent expression in control cells which have notbeen contacted with the stimulating agent and which have been contactedwith the candidate compound (control cells which have been contactedwith candidate compound alone) relative to CREB-dependent geneexpression in control cells which have been contacted with neither thestimulating agent or the candidate compound (control cells which havebeen contacted with nothing). In a particular embodiment, cells arecontacted with the candidate compound prior to contact with thestimulating agent.

Alternatively, the secondary screen comprises: (a) contacting cells ofneural origin comprising an indicator gene operably linked to a CREpromoter with a candidate compound identified in the primary screen andwith a suboptimal dose of a stimulating agent that activates signalingpathways onto CREB; (b) determining indicator activity in the cellswhich have been contacted with the candidate compound and with thestimulating agent; and (c) comparing indicator activity assessed in step(b) with indicator activity in control cells which have been contactedwith the stimulating agent and which have not been contacted with thetest compound (control cells which have been contacted with stimulatingagent alone). A statistically significant difference in indicatoractivity determined in step (b) compared to the indicator activity incontrol cells identifies the candidate compound as a confirmed candidatecompound. Preferably, no significant difference is obtained in indicatoractivity in control cells which have not been contacted with thestimulating agent and which have been contacted with the candidatecompound (control cells which have been contacted with candidatecompound alone) relative to indicator activity in control cells whichhave been contacted with neither the stimulating agent or the candidatecompound (control cells which have been contacted with nothing). In aparticular embodiment, cells are contacted with the candidate compoundprior to contact with the stimulating agent.

Preferably, the “stimulating agent that activates signaling pathwaysonto CREB” used in the primary screen is a CREB function stimulatingagent.

In another embodiment, the secondary screen can be replaced with ascreening method using Drosophila, wherein said screening methodcomprises: (a) administering a candidate compound identified in theprimary screen to Drosophila having an indicator gene operably linked toa CRE promoter, (b) assessing indicator activity in the Drosophila whichhave been administered the candidate compound; and (c) comparing theindicator activity assessed in step (b) with the indicator activity incontrol Drosophila which have not been administered the candidatecompound. A statistically significant difference in indicator activityin step (b) compared to the indicator activity in control Drosophilawhich have not been administered the candidate compound identifies thecandidate compound as a confirmed candidate compound.

In a particular embodiment, the cells used in the secondary screen aredifferent from the host cells used in the primary screen. In anotherembodiment, the endogenous CRE-mediated gene or the CRE-mediatedtransgene in the secondary screen is different from the CRE-mediatedtransgene or the endogenous CRE-mediated gene in the primary screen. Forexample, in one embodiment, the host cells in the primary screen areneuroblastomas and the cells for the secondary screen are notneuroblastomas. In another embodiment, the CRE-mediated transgene in theprimary screen is luciferase (CRE operably linked to the luciferasegene) and the CRE-mediated transgene in the secondary screen is notluciferase.

Preferably, the host cells in the primary screen are proliferating cells(such as neuroblastomas) and the cells in the secondary screen arenonproliferating, differentiated cells of neural origin (such as neurons(e.g., primary hippocampal cells) and glial cells). In a particularembodiment, the CRE-mediated gene in the primary screen is aCRE-mediated indicator gene (a CRE-mediated transgene) and theCRE-mediated gene in the secondary screen is an endogenous CRE-mediatedgene.

As used herein, a cell refers to an animal cell. The cell can be a stemcell or somatic cell. Suitable animal cells can be of, for example,mammalian origin. Examples of mammalian cells include human (such asHeLa cells), bovine, ovine, porcine, rodent (such as rat, murine (suchas embryonic stem cells), rabbit etc.) and monkey (such as COS1 cells)cells. Preferably, the cell is of neural origin (such as aneuroblastoma, neuron, neural stem cell, glial cell, etc.). The cell canalso be an embryonic cell, bone marrow stem cell or other progenitorcell. Where the cell is a somatic cell, the cell can be, for example, anepithelial cell, fibroblast, smooth muscle cell, blood cell (including ahematopoietic cell, red blood cell, T-cell, B-cell, etc.), tumor cell,cardiac muscle cell, macrophage, dendritic cell, neuronal cell (e.g., aglial cell or astrocyte), or pathogen-infected cell (e.g., thoseinfected by bacteria, viruses, virusoids, parasites, or prions). Thecells can be obtained commercially or from a depository or obtaineddirectly from an animal, such as by biopsy.

In one embodiment, the tertiary screen is a behavioral method forassessing long term memory formation in an animal comprising: (a)administering an effective amount of a confirmed candidate compoundidentified in the secondary screen to the animal (e.g., human, othermammal, vertebrate or invertebrate); (b) training the animaladministered the confirmed candidate compound under conditionsappropriate to produce long term memory formation in the animal; (c)assessing long term memory formation in the animal trained in step (b);and (d) comparing long term memory formation assessed in step (c) withlong term memory formation produced in the control animal to which theconfirmed candidate compound has not been administered. If anenhancement is noted in long term memory formation assessed in theanimal treated with the confirmed candidate compound relative to thelong term memory formation assessed in the control animal the confirmedcandidate compound is identified as a cognitive enhancer. Tertiaryscreens with similar protocols are available using behavioral methods(models) for other, cognitive dysfunctions.

In a particular embodiment, the method for identifying or screening forcognitive enhancers comprise: (a) contacting host cells comprising aluciferase reporter gene operably linked to a CRE promoter with a testcompound, thereby producing a test sample; (b) contacting the testsample produced in step (a) with a suboptimal dose of a CREB functionstimulating agent (e.g., forskolin, potassium chloride); (c) determiningCRE-luciferase activity in the host cells which have been contacted withthe test compound and with the CREB function stimulating agent; (d)comparing the CRE-luciferase activity determined in step (c) with theCRE-luciferase activity in control cells which have been contacted withthe CREB function stimulating agent and which have not been contactedwith the test compound (control cells which have been contacted withCREB function stimulating agent alone); (e) selecting the test compoundif it has little or no effect alone and if it further increasesCRE-luciferase levels (activity) in host cells which have been contactedwith the CREB function stimulating agent, thereby selecting an “ActiveHit” compound; (f) repeating steps (a) to (e) using a range of differentconcentrations of the Active Hit compound selected in step (e); and (g)selecting the Active Hit compound if it has little or no effect aloneand if it further proportionally changes the CRE-luciferase levels(activity) in host cells which have been contacted with the CREBfunction stimulating agent, for the linear range of concentrations ofthe Active Hit compound, thereby selecting a “Confirmed Active”compound. Host cells comprising a luciferase reporter gene operablylinked to a CRE promoter (CRE-driven luciferase reporter gene) can begenerated by transfecting the cells with a CRE-driven luciferasereporter gene. Such cells are also referred to herein as CRE-luci cells.In a particular embodiment, the host cells are human neuroblastomacells. Human neuroblastoma cells transfected with a CRE-drivenluciferase reporter gene are also referred to herein as CRElucineuroblastoma cells. A Confirmed Active Hit compound is also referred toherein as a candidate compound.

A Confirmed Active compound (or a candidate compound) can be assessed orevaluated for its effect on endogenous, CRE-mediated gene expression(endogenous CREB-dependent gene expression) by (a) contacting neurons(particularly hippocampal cells) with the Confirmed Active compound (orthe candidate compound), thereby producing a sample; (b) contacting thesample with a suboptimal dose of a CREB function stimulating agent; (c)assessing endogenous CREB-dependent gene expression in the neurons whichhave been contacted with the Confirmed Active compound (or candidatecompound) and the CREB function stimulating agent; and (d) comparingendogenous CREB-dependent gene expression assessed in step (c) withendogenous CREB-dependent gene expression in control neurons which havebeen contacted with the CREB function stimulating agent and which havenot been contacted with the Confirmed Active compound (or candidatecompound). A statistically significant difference in CREB-dependent geneexpression assessed in step (c) compared to the CREB-dependent geneexpression in control cells identifies the Confirmed Active compound (orcandidate compound) as having an effect on CREB-dependent geneexpression and as a confirmed candidate compound. Preferably, nosignificant difference is obtained in CREB-dependent expression incontrol cells which have not been contacted with the CREB functionstimulating agent and which have been contacted with the ConfirmedActive compound (or candidate compound) (control cells which have beencontacted with Confirmed Active compound (or candidate compound) alone)relative to CREB-dependent gene expression in control cells which havebeen contacted with neither the CREB function stimulating agent or theConfirmed Active compound (candidate compound) (control cells which havebeen contacted with nothing).

The confirmed candidate compound can be assessed or evaluated in ananimal (behavioral) model of CREB-dependent long-term memory formationto identify cognitive enhancers. Such methods for assessing orevaluating a confirmed candidate compound to identify cognitiveenhancers comprise (a) administering an effective amount of a confirmedcandidate compound to an animal; (b) training the animal administeredthe confirmed candidate compound under conditions appropriate to producelong term memory formation in the animal; (c) assessing long term memoryformation in the animal trained in step (b); and (d) comparing long termmemory formation assessed in step (c) with long term memory formationproduced in the control animal to which the confirmed candidate compoundhas not been administered. If a statistically significant enhancement isnoted in long term memory formation assessed in the animal treated withthe confirmed candidate compound relative to the long term memoryformation assessed in the control animal, the confirmed candidatecompound can be categorized as enhancing (improving, increasing) longterm memory formation in the animal and is identified as a cognitiveenhancer.

As described herein, confirmed candidate compounds and cognitiveenhancers from several chemical classes are progressed through in vivomodels of memory formation.

Active compounds are sought that do not enhance CREB pathway function ontheir own but rather after co-stimulation with a CREB functionstimulating agent. This requirement was introduced to mimic results fromthe original fly experiments: (1) cAMP signaling is involved in the flymemory formation and (2) training is required to enhance memoryformation when CREB activator is overexpressed.

Compounds to be evaluated or assessed for their ability to increase CREBpathway function, such as pharmaceutical agents, drugs, chemicalcompounds, ionic compounds, organic compounds, organic ligands,including cofactors, saccharides, recombinant and synthetic peptides,proteins, peptoids, nucleic acid sequences, including genes, nucleicacid products, and other molecules and compositions, can be individuallyscreened or one or more compound(s) can be tested simultaneously for theability to increase CREB pathway function in accordance with the methodsherein. Where a mixture of compounds is tested, the compounds selectedby the methods described can be separated (as appropriate) andidentified by suitable methods (e.g., chromatography, sequencing, PCR).The presence of one or more compounds in a test sample having theability to increase CREB pathway function can also be determinedaccording to these methods. Compounds to be screened for their abilityto increase CREB pathway function are generally at a concentration fromabout 10⁻⁹ molar to about 10⁻³ molar.

Large combinatorial libraries of compounds (e.g., organic compounds,recombinant or synthetic peptides, peptoids, nucleic acids) produced bycombinatorial chemical synthesis or other methods can be tested (seee.g., Zuckerman, R. N. et al., J. Med. Chem., 37:2678-2685 (1994) andreferences cited therein; see also, Ohlmeyer, M. H. J. et al., Proc.Natl. Acad. Sci. USA, 90:10922-10926 (1993) and DeWitt, S. H. et al.,Proc. Natl. Acad. Sci. USA, 90:6909-6913 (1993), relating to taggedcompounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D.et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). The teachings of these references are incorporated herein byreference. Where compounds selected from a combinatorial library carryunique tags, identification of individual compounds by chromatographicmethods is possible.

Chemical libraries, microbial broths and phage display libraries canalso be tested (screened) for the presence of one or more compoundswhich is capable of enhancing CREB pathway function in accordance withthe methods herein.

The invention also relates to methods for assessing the effect of acandidate compound on CREB-dependent gene expression (CRE-mediated geneexpression) comprising: (a) contacting cells of neural origin with thecandidate compound and with a suboptimal dose of a CREB functionstimulating agent; (b) assessing endogenous CREB-dependent geneexpression in the cells which have been contacted with the candidatecompound and with the CREB function stimulating agent; and (c) comparingendogenous CREB-dependent gene expression assessed in step (b) withendogenous CREB-dependent gene expression in control cells which havebeen contacted with the CREB function stimulating agent and which havenot been contacted with the candidate compound. A statisticallysignificant difference in CREB-dependent gene expression assessed instep (b) compared to the CREB-dependent gene expression in control cellsidentifies the candidate compound has one having an effect onCREB-dependent gene expression. Preferably, no significant difference isobtained in CREB-dependent expression in control cells which have notbeen contacted with the CREB function stimulating agent and which havebeen contacted with the candidate compound relative to CREB-dependentgene expression in control cells which have been contacted with neitherthe CREB function stimulating agent or the candidate compound (controlcells which have been contacted with nothing). In a particularembodiment, cells are contacted with the test compound prior to contactwith the CREB function stimulating agent.

Alternatively, methods for assessing the effect of a candidate compoundon CREB-dependent gene expression comprise: (a) contacting cells ofneural origin comprising an indicator gene operably linked to a CREpromoter with the candidate compound and with a suboptimal dose of aCREB function stimulating agent; (b) determining indicator activity inthe cells which have been contacted with the candidate compound and withthe CREB function stimulating agent; and (c) comparing indicatoractivity determined in step (b) with indicator activity in control cellswhich have been contacted with the CREB function stimulating agent andwhich have not been contacted with the candidate compound. Astatistically significant difference in indicator activity determined instep (b) compared to the indicator activity in control cells identifiesthe candidate compound has one having an effect on CREB-dependent geneexpression. Preferably, no significant difference is obtained inindicator activity in control cells which have not been contacted withthe CREB function stimulating agent and which have been contacted withthe candidate compound relative to indicator activity in control cellswhich have been contacted with neither the CREB function stimulatingagent or the candidate compound (control cells which have been contactedwith nothing). In a particular embodiment, cells are contacted with thetest compound prior to contact with the CREB function stimulating agent.

As with the secondary screen, in a particular embodiment, the cells usedin the methods for assessing the effect of a candidate compound onCREB-dependent gene expression are different from the host cells in theprimary screen. In another embodiment, the endogenous CRE-mediated geneor the CRE-mediated transgene used in the methods for assessing theeffect of a candidate compound on CREB-dependent gene expression aredifferent from the CRE-mediated transgene or the endogenous CRE-mediatedgene in the primary screen. In another embodiment, the cells used in themethods for assessing the effect of a candidate compound onCREB-dependent gene expression are nonproliferating, differentiatedcells of neural origin (such as neurons (e.g., primary hippocampalcells) and glial cells). In a particular embodiment, the CRE-mediatedgene in the methods for assessing the effect of a candidate compound onCREB-dependent gene expression is an endogenous CRE-mediated gene.

Enhancement of long term memory formation can be defined as (a) anincrease in levels of behavioral performance; (b) a decrease in thenumber of training trials required to produce a level of behavioralperformance; or (c) a decrease in the duration of rest between trainingsessions to produce a level of behavioral performance. Accordingly, theinvention also relates to methods for assessing the effect of acognitive enhancer on long term memory formation in an animalcomprising: (a) administering an effective amount of cognitive enhancerto an animal; (b) training the animal administered the cognitiveenhancer under conditions appropriate to produce long term memoryformation in said animal; (c) assessing long term memory formation inthe animal trained in step (b); and (d) comparing long term memoryformation assessed in step (c) with long term memory formation producedin the control animal to which the cognitive enhancer has not beenadministered. If an enhancement is noted in long term memory formationassessed in the animal treated with the cognitive enhancer relative tothe long term memory formation assessed in the control animal, thecognitive enhancer can be categorized as enhancing long term memoryformation in the animal. Cognitive enhancers identified as enhancinglong term memory formation in the animal are expected to be effectivecandidates for use in rehabilitating an animal with cognitivedysfunctions and for use in enhancing memory or normal cognitiveperformance (ability or function) in a normal animal. It is understoodthat, in certain instances, a cognitive enhancer identified inaccordance with the present invention can be a CREB function stimulatingagent Thus, a CREB function stimulating agent may be identified as acognitive enhancer in accordance with the invention herein.

The phrases “behavior performance” and “cognitive performance” areart-recognized phrases and are used herein in accordance with theirart-accepted meaning.

As used herein, the term “animal” includes mammals, as well as otheranimals, vertebrate and invertebrate (e.g., birds, fish, reptiles,insects (e.g., Drosophila species), mollusks (e.g., Aplysia). The terms“mammal” and “mammalian”, as used herein, refer to any vertebrateanimal, including monotremes, marsupials and placental, that suckletheir young and either give birth to living young (eutharian orplacental mammals) or are egg-laying (metatharian or nonplacentalmammals). Examples of mammalian species include humans and primates(e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs)and ruminents (e.g., cows, pigs, horses).

The animal can be an animal with some form and degree of cognitivedysfunction or an animal with normal cognitive performance (i.e., ananimal without any form of cognitive failure (dysfunction or loss of anycognitive function)).

An effective amount of cognitive enhancer or confirmed candidatecompound is that amount, or dose, administered to an animal that isrequired to effect a change (increase or decrease) in CREB-dependentgene expression, particularly in cells of neural origin. The dosageadministered to an animal, including frequency of administration, willvary depending upon a variety of factors, including pharmacodynamic andpharmacokinetic characteristics of the particular cognitive enhancer orconfirmed candidate compound, mode and route of administration; size,age, sex, health, body weight and diet of the recipient; nature andextent of symptoms being treated or nature and extent of the cognitivedysfunction(s) being enhanced or modilated, kind of concurrenttreatment, frequency of treatment, and the effect desired.

Training of animals for long term memory formation are conducted usingmethods generally known in the art (see, e.g., Josselyn et al., Societyfor Neurosci., 24:926, Abstract 365.10 (1998); Casella and Davis,Physiol. Behav., 36:377-383 (1986); Guzowsli et al., Proc. Natl. Acad.Sci. USA, 94:2693-2698 (1997); Lamprecht et al., J. Neuroscience,17(21):6443-6450 (1997): Bourtchuladze et al., Cell, 79:59-68 (1994);Kogan et al., Curr. Biol., 7:1-11 (1996); Tully and Quinn, J. Comp.Physiol. A Sens. Neural. Behav. Physiol., 157:263-277 (1985); Tully etal., Cell, 79:35-47 (1994)). Training can comprise one or multipletraining sessions. By “multiple training sessions” is meant two or moretraining sessions.

The invention further relates to methods for assessing the effect of acognitive enhancer on olfactory memory formation in Drosophilacomprising: (a) administering an effective amount of cognitive enhancerto Drosophila; (b) subjecting the Drosophila to classical conditioningand to at least one odorant and electrical shock; and (c) assessing theperformance index of the classical conditioning, wherein the effect ofthe cognitive enhancer occurs when the compound alters the performanceindex from the performance index obtained by the Drosophila of step (a)in the absence of the cognitive enhancer.

The invention also relates to each of the primary, secondary andtertiary screens comprising the methods for identifying or screening forcognitive enhancers described herein.

The invention anticipates a new type of cognitive enhancer that enhancesCREB pathway function and augments the therapeutic effects of cognitivetraining. Such cognitive enhancers can be used as a general therapy forvarious forms of cognitive dysfunction arising from heredity, disease,injury and age by stimulating the molecular process contributing tobrain plasticity and for enhancing memory or normal cognitiveperformance (ability or function).

Cognitive enhancers identified in accordance with the methods of theinvention are compounds with pharmacological activity and include drugs,chemical compounds, ionic compounds, organic compounds, organic ligands,including cofactors, saccharides, recombinant and synthetic peptides,proteins, peptoids, nucleic acid sequences, including genes, nucleicacid products, and other molecules and compositions.

By “modulate” is meant the include the ability to induce, enhance,potentiate, reduce, block, inhibit (total or partial) and regulate abiochemical or physiological action or effect. By “regulate”, as theterm is used herein, is meant the ability to control the rate and extentto which a process occurs. By “enhance” or “enhancing” is meant theability to potentiate, increase, improve or make greater or better,relative to normal, a biochemical or physiological action or effect.

Cognitive dysfunction, commonly associated with brain dysfunction andcentral nervous system (CNS) disorders or conditions, arises due toheredity, disease, injury and/or age. CNS disorders and conditionsassociated with some form and degree of cognitive failure (dysfunction)include, but are not limited to the following:

-   -   1) age-associated or age-dependent memory impairment;    -   2) mental retardation arising due to a heritable defect, such        as, but not limited to that associated with (due to),        chromosomal abnormalities, including Rubinstein-Taybi syndrome,        down syndrome, fragile X syndrome, cri du chat syndrome,        Klinefelter's syndrome, Turner's syndrome, mosaicisms, Patau's        syndrome (trisomy 13) and Edward's syndrome (trisomy 18);        genetic metabolic abnormalities, including Coffin-Lowry        syndrome, autosomal recessive disorders, such as aminoacidurias        and acidemias, peroxisomal disorders, such as galactosemia,        maple syrup urine disease and phenylketonuria, lysosomal        defects, such as Gaucher's disease, Hurler's syndrome        (mucopolysaccharidosis), Niemann-Pick disease and Tay-Sachs        disease, and X-linked recessive disorders, such as Lesch-Nyhan        syndrome (hyperuricemia), Hunter's syndrome (a variant of        mucopolysaccharidosis) and Lowe's oculocerebrorenal syndrome;        genetic neurological abnormalities, including autosomal dominant        disorders, such as myotonic dystrophy, neurofibromatosis and        tuberous sclerosis, and autosomal recessive disorders, such as        primary microcephaly,    -   3) trauma-dependent loss of cognitive function, such as, but not        limited to that associated with (due to), cerebrovascular        diseases, including stroke and ischemia, including ischemic        stroke; brain trauma, including subdural hematoma and brain        tumor; head injury;    -   4) neurodegenerative disorders, such as delirium (acute        confusional state); dementia, including Alzheimer's disease and        non-Alzheimer's type dementias, such as, but not limited to,        Lewy body dementia, vascular dementia, Binswanger's dementia        (subcortical arteriosclerotic encephalopathy), dementias        associated with Parkinson's disease, progressive supranuclear        palsy, Huntington's disease (chorea), Pick's disease,        normal-pressure hydrocephalus, Creutzfeldt-Jakob disease,        Gerstmann-Sträussler-Scheinker disease, neurosyphilis (general        paresis) or HIV infection, frontal lobe dementia syndromes,        dementias associated with head trauma, including dementia        pugilistica, brain trauma, subdural hematoma, brain tumor,        hypothyroidism, vitamin B₁₂ deficiency intracranial radiation;        other neurodegenerative diseases and disorders;    -   5) psychiatric disorders, including affective disorders (mood        disorders), such as, but not limited to, depression, including        depressive pseudodementia; psychotic disorders, such as, but not        limited to, schizophrenia and autism (Kanner's Syndrome);        neurotic disorders, such as, but not limited to, anxiety and        obsessive-compulsive disorder; attention deficit disorder; and    -   6) learning, language or reading disabilities, particularly in        children. By “learning disabilities” is meant disorders of the        basic psychological processes that affect the way an individual        learns. Learning disabilities can cause difficulties in        listening, thinking, talking, reading, writing, spelling,        arithmetic or combinations of any of the foregoing. Learning        disabilities include perceptual handicaps, dyslexia and        developmental aphasia.

Cognitive enhancers and confirmed candidate compounds can beadministered directly to an animal in a variety of ways. In a preferredembodiment, cognitive enhancers and confirmed candidate compounds areadministered systemically. Other routes of administration are generallyknown in the art and include intravenous including infusion and/or bolusinjection, intracerebroventricularly, intrathecal, parenteral, mucosal,implant, intraperitoneal, oral, intradermal, transdermal (e.g., in slowrelease polymers), intramuscular, subcutaneous, topical, epidural, etc.routes. Other suitable routes of administration can also be used, forexample, to achieve absorption through epithelial or mucocutaneouslinings. Particular cognitive enhancers and confirmed candidatecompounds can also be administered by gene therapy, wherein a DNAmolecule encoding a particular therapeutic protein or peptide isadministered to the animal, e.g., via a vector, which causes theparticular protein or peptide to be expressed and secreted attherapeutic levels in vivo.

A vector, as the term is used herein, refers to a nucleic acid vector,e.g., a DNA plasmid, virus or other suitable replicon (e.g., viralvector). Viral vectors include retrovinis, adenovims, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovinis (e.g. measles and Sendai),positive strand RNA viruses such as picomavirus and alphavinis, anddouble stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996). Other examples include murine leukemiaviruses, murine sarcoma viruses, mouse mammary tumor virus, bovineleukemia virus, feline leukemia virus, feline sarcoma virus, avianleukemia virus, human T-cell leukemia virus, baboon endogenous virus,Gibbon ape leukemia virus, Mason Pfizer monkey virus, simianimmunodeficiency virus, simian sarcoma virus, Rous sarcoma virus andlentiviruses. Other examples of vectors are described, for example, inMcVey et al., U.S. Pat. No. 5,801,030, the teachings of which areincorporated herein by reference.

The mode of administration is preferably at the location of the targetcells. In a particular embodiment, the mode of administration is tocells of neural origin Cells of neural origin include neural stem cells,neuroblastoma cells, neurons and glial cells.

Cognitive enhancers and confirmed candidate compounds can beadministered together with other components of biologically activeagents, such as pharmaceutically acceptable surfactants (e.g.,glycerides), excipients (e.g., lactose), stabilizers, preservatives,humectants, emollients, antioxidants, carriers, diluents and vehicles.If desired, certain sweetening, flavoring and/or coloring agents canalso be added.

Cognitive enhancers and confirmed candidate compounds can be formulatedas a solution, suspension, emulsion or lyophilized powder in associationwith a pharmaceutically acceptable parenteral vehicle. Examples of suchvehicles are water, saline, Ringer's solution, isotonic sodium chloridesolution, dextrose solution, and 5% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation can be sterilized by commonly usedtechniques. Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences.

Cognitive enhancers and confirmed candidate compounds can beadministered in single or divided doses (e.g., a series of dosesseparated by intervals of days, weeks or months), or in a sustainedrelease form, depending upon factors such as nature and extent ofsymptoms, kind of concurrent treatment and the effect desired. Othertherapeutic regimens or agents can be used in conjunction with thepresent invention.

The present invention will now be illustrated by the following example,which is not to be considered limiting in any way.

EXAMPLES Example 1

From the drug discovery program described herein, lead compounds fromseveral chemical classes are progressed through in vivo models of memoryformation.

Drug Discovery

Two basic principles guide the drug discovery program. First, activecompounds are sought that do not enhance CREB function on their own butrather after co-stimulation with forskolin, an activator of adenylylcyclase. This requirement was introduced to mimic results from theoriginal Drosophila experiments: (1) cAMP signaling is involved in thefly memory formation and (2) training is required to enhance memoryformation when CREB activator is overexpressed. Second, CREB appears tofunction similarly in neurons from various vertebrate and invertebratespecies. To capitalize on this evolutionary conservation, a drugprogression pathway was developed that includes fly, rodent and humanassays.

Primary Screen

A cell-based, high throughput screen (HTS) was designed to assess theeffects of compounds on CREB pathway function. Human neuroblastoma cells(SKN-MC) were transfected stably with a reporter construct, in whichluciferase was placed under the control of a CRE promoter. With thisreporter, increases in CREB pathway function were monitored viafluorescent output. This tester cell line was evaluated fully usingknown CREB activators.

To assess compound activity, the tester line was pre-incubated in thepresence of compound and then was stimulated with a suboptimal dose offorskolin prior to lysis. The concentration and time-course forforskolin co-stimulation were calibrated to reproducibly obtain 30%saturation, yielding a 300% range for compound effects. Compounds thatproduced ≧100% increase in luciferase signal over control cellsstimulated with forskolin alone were progressed to a second round ofscreening to exclude statistical false positives. Finally, thesecompounds were assessed at four different concentrations with or withoutforskolin co-stimulation.

To date, 155,000 compounds have been screened with this approach,yielding about 180 Confirmed Actives.

Secondary Screens

Eighty six of the 180 Confirmed Actives were chosen for further analysisbased on their structure and potency. To rule out any potentialartifactual effects of the reporter gene construct, real timequantitative PCR (QPCR) was used to assess expression levels of known,endogenous CREB-dependent genes in the tester line. Most of theconfirmed actives showed comparable potency profiles for both luciferasereporter gene expression and endogenous gene expression. Structuralanalyses of these 86 Confirmed Actives revealed 10 to be digitoxigeninrelated, 26 to be phenylethylamine derivatives, 4 to be aminopropanediolderivatives, 3 to be phosphodiesterase inhibitors and 3 to be caffeineanalogues. The remaining 40 were novel chemical structures with noobvious similarities to other known compounds. Of these, 11 Active Hitswere chosen for further study based on potency, solubility and ease toderivatize. For these 11 Active Hits, enhancement of endogenousCREB-dependent gene expression was confirmed in cultured rat primaryhippocampal neurons. Functional (enzymatic, binding affinity, etc.)analyses revealed 8 to be PDE IV inhibitors and 2 to target othermolecules.

In Vivo Assays

The 11 Active Hits were fed to transgenic flies, carrying aCRE-luciferase transgene. Previous experiments had established that: (1)reporter gene activity could be quantified from single flies and (2) theCRE-luciferase reporter normally showed a circadian cycle of expression(Belvin, M. P. et al., Neuron, 22(4):777-787 (1999)). These observationsrevealed an efficient initial in vivo assay with which to determine thedrug concentrations needed to affect CRE-luciferase expression in braintissue of drug-fed flies. All 11 Lead Candidates produced a significantincrease in CRE-luciferase expression levels in vivo. One of thesecompounds was tested in vivo for its effects on fly olfactory memoryformation. It showed a partial enhancement of memory after massedtraining and no effect on memory after spaced training-a resultreminiscent of the CREB signature.

These Active Hits were evaluated in vivo rodent models of memoryformation. To do so efficiently, a new behavioral training protocol wasvalidated, which logically was affected by upstream modulation of theCREB pathway. During initial stages of acquisition, repeated trainingtrials lead to increased cAMP levels. At some point, cAMP levels pass athreshold, and activated PKA (catalytic subunit) is translocated to thenucleus, where it phosphorylates CREB directly or it yields a permissivestate for the phosphorylation of CREB by a cofactor. In this context,phosphodiesterase (PDE) inhibitors act to increase cAMP levels beyondthe threshold with fewer training trials, thereby producing morelong-lasting memory with weaker training. Using the prototypical PDEinhibitor, rolipram, this effect was established as predicted (FIG. 1).

Taken together these data indicate that PDE is an efficacious drugtarget to enhance CREB function.

Example 2

Method for Identifying Functional CREB Antisense Oligos.

Detection of CREB mRNA with the bDNA Assay.

Mus musculus Neuro2a cells were obtained from American Type CultureCollection (ATCC, Cat. No. CCL-131; Manassas, Va.). Cells were plated inModified Eagle's Minimum Essential Media (Minimum essential medium(Eagle) with 2 mM L-glutamine and Earle's BSS adjusted to contain 1.5g/L sodium bicarbonate, 0.1 mM non-essential amino acids, and 1.0 mMsodium pyruvate, 90%; fetal bovine serum, 100%) in a 96-microwell formatat 2×10⁴ cells per well. Cells were maintained at 37° C. and 5% CO₂overnight before transfecting with CREB Locked Nucleic Acid (LNA)oligonucleotides.

Cells were transfected using Lipofectamine 2000 (hivitrogen, Carlsbad,Calif.) with modifications to the manufacturer's protocol. For eachtransfection sample, the corresponding CREB LNA oligonucleotide (seesequences below) was diluted with 25 μl Opti-MEM reduced-serum media(HEPES buffer, 2,400 mg/L sodium bicarbonate, hypoxanthine, thyrmidine,sodium pyruvate, L-glutamine, trace elements, growth factors, and phenolred reduced to 1.1 mg/L (Invitrogen) to a final transfectionconcentration of 0.2 μM. The Lipofectamine 2000 was also diluted in 25μl Opti-MEM reduced-serum media so the ratio of LNA oligonucleotide (μl)to Lipofectamine 2000 (μl) was 1:3, respectively. The diluted LNAoligonucleotide was combined with the diluted Lipofectamine 2000 andincubated for 15 minutes at room temperature for the complexes to form.Fifty μl of LNA oligonucleotide-Lipofectamine 2000 complexes were addedto each well. Transfections were performed in triplicate for each CREBLNA oligonucleotide at 0.2 μM. The cells were incubated at 37° C. and 5%CO₂ for 24 hours. Growth medium was replaced after 5 hours with 100 μlMEME medium.

The direct quantitation of CREB messenger RNA (mRNA) followingtransfection of antisense oligos, was detected using the QuantiGeneExpression Kit according to the manufacturer's protocol (BayerCorporation; Tarrytown, N.Y.). Twenty four hours post antisense LNAoligonucleotide transfections, the cells were lysed with 50 μl lysisbuffer (Bayer Corporation) containing the pooled CREB specific CaptureExtenders (CE), Label Extenders (LE), and blocking (BL) oligonucleotides(described below). After a 30 minute incubation, 100 μl of the lysismixture from each well were added to a corresponding capture well (BayerCorporation). The capture plate was sealed with an adhesive-backed PlateSealer (Bayer Corporation) and incubated overnight at 50° C. The wellswere washed three times with 390 μl of wash buffer (0.1×SSC and 0.03%Lithium Lauryl Sulfate) in an ELx405 Plate Washer (BioTek Instruments).100 μl of amplifier reagent containing amplifier probe diluted 1:1000 inamplifier/label probe diluent (provided by Bayer Corporation) wasimmediately added to each well of the capture plate, sealed, andincubated at 50° C. for 60 minutes. The wells were washed three timeswith wash buffer as described above and 100 μl of labeling reagentcontaining label probe diluted 1:1000 in amplifier/label probe diluent(Bayer Corporation) was immediately added to each well of the captureplate. The capture plate was sealed and incubated at 50° C. for 60minutes. The wells were washed three times with wash buffer as describedabove and 100 μl of substrate working solution (0.003% Lithium LaurylSulfate in Substrate Working Reagent; Bayer Corporation) was immediatelyadded to each well of the capture plate. The capture plate was sealedand incubated at 50° C. for 30 minutes. The luminescence of each wellwas measured in a Wallac Victor² V (Perkin Elmer) plate luminometer at45° C.

FIG. 3 shows the effects of various different CREB antisense LNA oligoson levels of CREB RNA in Neuro2a 131 cells following transfection,relative to cells treated with vehicle control (transfection reagentminus any oligo). Each CREB LNA oligo has a unique sequence (see below).The CREB LNB oligos were numbered in the order that they were designed.CREB LNB oligos that were not screened (tested) because they did nothave suitable properties (e.g., melting temperature) when analyzed(i.e., CREB 4, 10, 11, 13 and 14) are not shown.

The results demonstrate that oligos CREB1 and CREB17 reduce endogenousCREB RNA levels by at least 50%.

Oligo CREB3 is a sequence that has previously been used to knock downCREB RNA (as a phosphorothioate oligo). The results herein demonstratethat oligo CREB3 also reduces endogenous CREB RNA levels, although at ahigher concentration (0.5 μM).

CREB Locked Nucleic Acid Oligonucleotide Design.

Antisense oligonucleotides were designed for CREB using the sequence forGenBank Accession Number M95106 with Vector NTI Suite 8 (InforMax,Frederick, Md.). The sequences of the Locked Nucleic Acid (LNA)oligonucleotides (Proligo LLC, Boulder, Colo.) are summarized below: 1.CCTCCgCCgCgTCACTCA (SEQ ID NO.1) 2. CCACgTAACACACCgCgT (SEQ ID NO.:2) 3.TggTCATTTAgTTACCggTg (SEQ ID NO.:3) 4. gCTggTTgTCTgCACCAG (SEQ ID NO.:4)5. TTTTCAgCTTCTgTTACA (SEQ ID NO. 5) 6. CTgggCTTgAACTgTCAT (SEQ IDNO.:6) 7. gCTAATgTggCAATCTgT (SEQ ID NO.:7) 8. TgCTggCATggATACCTg (SEQID NO.:8) 9. gCAgATgATgTTgCATgA (SEQ ID NO.:9) 10. TgTCTgCCCATTgggCAg(SEQ ID NO.:10) 11. gggCCgCCTggATAACgCC (SEQ ID NO.:11) 12.TTCACTTTCTgCAATAgT (SEQ ID NO.:12) 13. TCCACAgACTCCTgTgAA (SEQ IDNO.:13) 14. AAAggATTTCCCTTCgTT (SEQ ID NO.:14) 15. CAgAAgATAAgTCATTCA(SEQ ID NO.:15) 16. TTCTCAATCCTTggCAC (SEQ ID NO.:16) 17.TggCACTgTTACAgTggT (SEQ ID NO.:17) 18. CTgCCCACTgCTAgTTTg (SEQ IDNO.:18) 19. gCTCCTCCgTCACTgCTT (SEQ ID NO.:19) 20. TgCACTAAggTTACAgTg.(SEQ ID NO.:20)

Individual LNA oligonucleotides were resuspended in 1×TE (10 mM TrisHCl, pH 8.0 and 1 mM EDTA, pH 8.0) at a concentration of 200 μM.

CREB bDNA Probe Design.

Oligonucleotide probes were designed for the capture and signalamplification of the CREB mRNA with QuantiGene ProbeDesigner Software(Bayer Corporation). The sequences of oligonucleotides (Integrated DNATechnologies Inc; Coralville, Iowa) are summarized below: CEggatttcccttcgtttttgggTTTTTctcttggaaagaaagt (SEQ ID NO.:21) CEcaatccttggcaccctggtTTTTTctcttggaaagaaagt (SEQ ID NO.:22) CEagtctcctcttctgacttttcttcttTTTTTctcttggaaagaaagt (SEQ ID NO.:23) CEtcctccctgggtaatggcaTTTTTctcttggaaagaaagt (SEQ ID NO.:24) CEccattgttagccagctgtattgcTTTTTctcttggaaagaaagt (SEQ ID NO.:25) CEccggctgagtggcagctgTTTTTctcttggaaagaaagt (SEQ ID NO.:26) CEgcctgaggcagcttgaacaTTTTTctcttggaaagaaagt (SEQ ID NO.:27) CEtgctgcttcttcagcaggctTTTTTctcttggaaagaaagt (SEQ ID NO.:28) CEttagacggacctctctcttccgTTTTTctcttggaaagaaagt (SEQ ID NO.:29) LEgaatcttcactttctgcaatagttgaTTTTTaggcataggacccgtgtct (SEQ ID NO.:30) LEaatcagttacactatccacagactcctgtTTTTTaggcataggacccgtgtct (SEQ ID NO.:31) LEatgtactgcccactgctagtttggtaTTTTTaggcataggacccgtgtct (SEQ ID NO.:32) LEggccctgtaccccatccgtaTTTTTaggcataggacccgtgtct (SEQ ID NO.:33) LEcattggtcatggttaatgtctgcaTTTTTaggcataggacccgtgtct (SEQ ID NO.:34) LEgtctgtgcatactgtagaatggtagtacTTTTTaggcataggacccgtgtct (SEQ ID NO.:35) LEagaatctgctgtccatccgtgTTTTTaggcataggacccgtgtct (SEQ ID NO.:36) LEgtgcgaatctggtatgtttgtacatcTTTTTaggcataggacccgtgtct (SEQ ID NO.:37) LEacgccataacaactccagggTTTTTaggcataggacccgtgtct (SEQ ID NO.:38) BLcctgtaggaaggcctccttgaaa (SEQ ID NO.:39) BL gcatcagaagataatcattcaaaatttt(SEQ ID NO.:40) BL tggtgatggcaggggctga (SEQ ID NO.:41) BLaatgggggttggcactgttacag (SEQ ID NO.:42) BL acaacttggttgctgggcact (SEQ IDNO.:43) BL gcaatggtgctagtgggtgct (SEQ ID NO.:44) BLgtgtaggaagtgctggggagg (SEQ ID NO.:45)

Individual (CE), (LE), and (BL) probes were resuspended in 1×TE, pH 8.0at a concentration of 100 pmoles/μl. The target specific (CE), (LE), and(BL) probe set pools were resuspended in 1×TE at a final concentrationof 50, 200, and 100 fmol/μl, respectively.

Effect of CREB Antisense LNA oligos on CRE-Luci Response toForskolin+HT0712 in SK-N-MC Cells.

Human SK-N-MC cells (see Example 1), stably transfected with a reporterconstruct consisting of the VIP promoter containing 2 CRE elementstogether with 2 additional CRE elements from the tyrosineaminotransferase gene driving expression of luciferase, were plated inIscoves Modified Eagle's Medium (IMEM, Invitrogen, Carlsbad, Calif.),with 2 mM L-glutamine, 0.1 mM Non-essential amino acids, 0.1 mM HEPES,10% fetal bovine serum at a density of 20,000 cells per well in a 96well plate format. Cells were maintained at 37° C., 5% CO₂ overnightbefore transfection with 0.2 μM CREB LNA antisense oligonucleotides.Functional CREB oligos were identified in the bDNA screen describedearlier. CREB LNA oligos utilized were: CREB1: CCTCCgCCgCgTCACTCA (SEQID NO.:46) CREB 3: CCACgTAACACACCgCgT (SEQ ID NO.:47) CREB 17:TggCACTgTTACAgTggT (SEQ ID NO.:48)

CREB 1 and CREB 3 oligos have identical sequence homology with bothmouse and human. CREB 17 has a single base mismatch. All three LNAoligos have been shown to be factional in knocking down human CREB RNAlevels in a human CREB bDNA assay.

Cells were transfected with human CREB LNA oligos using Lipofectamine2000 (Invitrogen, Carlsbad, Calif.) with modifications to themanufacturer's protocol. For each transfection sample, the correspondingCREB LNA oligonucleotide (CREB oligos 1, 3 and 17) was diluted with 25μl Opti-MEM reduced-serum media (HEPES buffer, 2,400 mg/L sodiumbicarbonate, hypoxanthine, thymidine, sodium pyruvate, L-glutamine,trace elements, growth factors, and phenol red reduced to 1.1 mg/L;Invitrogen) to a final transfection concentration of 0.2 μM. TheLipofectamine 2000 was also diluted in 25 μl Opti-MEM reduced-serummedia so the ratio of LNA oligonucleotide (μl) to Lipofectamine 2000(μl) was 1:3, respectively. The diluted LNA oligonucleotide was combinedwith the diluted Lipofectamine 2000 and incubated for 15 minutes at roomtemperature for the complexes to form. 50 μl of LNAoligonucleotide-Lipofectamine 2000 complexes were added to each well.The cells were incubated at 37° C. and 5% CO₂ for 16-24 hours. Growthmedium was replaced after 5 hours with 100 μl IMEM medium.

5 μM HT0712 ((3S,5S)-5-(3-cyclopentyloxy-4-methoxy-phenyl)-3-(3-ethyl-benzyl)-piperidin-2-one;also known as IPL 455,903)) was added to the ransfected/vehicle treatedcells, and incubated at 37 C, 5% CO₂ for 2 hours. HT0712 has thefollowing formula:

wherein “Me” means “methyl” and “cPent” means “cyclopentyl”. HT0712 canbe prepared using the methodology provided in U.S. Pat. No. 6,458,829B1,the teachings of which are incorporated herein by reference.

2 μM forskolin was then added, and the cells incubated for a further 6hours, washed briefly with PBS, lysed in the presence of luciferin andluciferase activity measured using a Victor 5 luminometer.

The teachings of all the articles, patents and patent applications citedherein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for identifying candidate compounds for enhancing CREBpathway function comprising the steps of: a) contacting host cellscomprising an indicator gene operably linked to a CRE promoter with atest compound and with a suboptimal dose of a CREB function stimulatingagent; b) determining indicator activity in said host cells which havebeen contacted with said test compound and with said CREB functionstimulating agent; c) comparing the indicator activity determined instep c) with the indicator activity in control cells which have beencontacted with said CREB function stimulating agent and which have notbeen contacted with said test compound; d) selecting said test compoundif: i) the indicator activity determined in step b) is increasedrelative to the indicator activity in said control cells which have beencontacted with said CREB function stimulating agent and which have notbeen contacted with said test compound; and ii) the indicator activityin control cells which have not been contacted with said CREB functionstimulating agent and which have been contacted with said test compoundis not significantly different relative to the indicator activity incontrol cells which have not been contacted with said CREB functionstimulating agent and which have not been contacted with said testcompound; e) repeating steps a) to d) with a range of differentconcentrations of said test compound selected in step d); and f)selecting said test compound if: i) the indicator activity is increasedin the range of concentrations for said test compound relative to theindicator activity in said control cells which have been contacted withsaid CREB function stimulating agent and which have not been contactedwith said test compound; and ii) the indicator activity in control cellswhich have not been contacted with said CREB function stimulating agentand which have been introduced said range of different concentrations ofsaid test compound is not significantly different relative to theindicator activity in control cells which have not been contacted withsaid CREB function stimulating agent and which have not been contactedwith said test compound, wherein said test compound is identified as acandidate compound for enhancing CREB pathway function.
 2. The method ofclaim 1 wherein said host cells are contacted with said test compoundprior to contact with said CREB function stimulating agent.
 3. Themethod of claim 1 wherein said host cells are human neuroblastoma cells.4. The method of claim 1 wherein said indicator gene encodes luciferase.5. The method of claim 1 wherein said CREB function stimulating agent isforskolin.
 6. The method of claim 4 wherein steps a) to d) are repeatedwith a range of four different concentrations of said test compoundselected in step d).
 7. The method of claim 1 further comprising thesteps of: g) contacting cells of neural origin with said candidatecompound and with a suboptimal dose of a CREB function stimulatingagent, wherein said cells of neural origin are different from the hostcells of step a); h) assessing endogenous CREB-dependent gene expressionin said cells which have been contacted with said candidate compound andwith said CREB function stimulating agent; and i) comparing endogenousCREB-dependent gene expression assessed in step h) with endogenousCREB-dependent gene expression in control cells which have beencontacted with said CREB function stimulating agent and which have notbeen contacted with said candidate compound, wherein a difference inCREB-dependent gene expression assessed in step h) compared to theCREB-dependent gene expression in control cells confirms that saidcompound is a candidate compound for enhancing CREB pathway function,thereby identifying said candidate compound as a confirmed candidatecompound.
 8. The method of claim 7 wherein said cells of neural originare contacted with said candidate compound prior to contact with saidCREB function stimulating agent.
 9. The method of claim 7 wherein saidcells of neural origin are neurons.
 10. The method of claim 9 whereinsaid neurons are primary hippocampal cells.
 11. The method of claim 7wherein said CREB function stimulating agent is forskolin.
 12. A methodfor assessing the effect on CREB-dependent gene expression of acandidate compound for enhancing CREB pathway function comprising thesteps of: a) contacting cells of neural origin with a candidate compoundand with a suboptimal dose of a CREB function stimulating agent; b)assessing endogenous CREB-dependent gene expression in the cells whichhave been contacted with said candidate compound and with said CREBfunction stimulating agent; and c) comparing endogenous CREB-dependentgene expression assessed in step b) with endogenous CREB-dependent geneexpression in control cells which have been contacted with said CREBfunction stimulating agent and which have not been contacted with saidcandidate compound.
 13. The method of claim 12 wherein said cells ofneural origin are contacted with said candidate compound prior tocontact with said CREB function stimulating agent.
 14. The method ofclaim 12 wherein said cells of neural origin are neurons.
 15. The methodof claim 14 wherein said neurons are primary hippocampal cells.
 16. Themethod of claim 12 wherein said CREB function stimulating agent isforskolin.
 17. A method for assessing the effect on long term memoryformation in an animal of a candidate compound for enhancing CREBpathway function comprising the steps of: a) administering saidcandidate compound to be assessed to said animal; b) training saidanimal administered said compound under conditions appropriate toproduce long term memory formation in said animal; c) assessing longterm memory formation in said animal trained in step b); and d)comparing long term memory formation assessed in step c) with long termmemory formation produced in the control animal to which said candidatecompound has not been administered.
 18. The method of claim 17 whereinsaid animal is a mammal.
 19. A method for screening a compound for itsability to enhance CREB pathway function comprising the steps of: a)contacting host cells comprising an indicator gene operably linked to aCRE promoter with a test compound, thereby producing a test sample; b)contacting the test sample produced in step a) with a suboptimal dose ofa CREB function stimulating agent; c) determining indicator activity insaid host cells which have been contacted with said test compound andwith said CREB function stimulating agent; d) comparing the indicatoractivity determined in step c) with the indicator activity in controlcells which have been contacted with said CREB function stimulatingagent and which have not been contacted with said test compound; e)selecting said test compound if: 1) the indicator activity determined instep c) is increased relative to the indicator activity in said controlcells which have been contacted with said CREB function stimulatingagent and which have not been contacted with said test compound; and 2)the indicator activity in control cells which have not been contactedwith said CREB function stimulating agent and which have been contactedwith said test compound is not significantly different relative to theindicator activity in control cells which have not been contacted withsaid CREB function stimulating agent and which have not been contactedwith said test compound; f) repeating steps a) to e) with a range ofdifferent concentrations of said test compound selected in step e); g)selecting said test compound if: 1) the indicator activity is increasedin the range of different concentrations for said test compound relativeto the indicator activity in said control cells which have beencontacted with said CREB function stimulating agent and which have notbeen contacted with said test compound; and 2) the indicator activity incontrol cells to which have not been contacted with said CREB functionstimulating agent and which have been introduced said range of differentconcentrations of said test compound is not significantly differentrelative to the indicator activity in control cells which have not beencontacted with said CREB pathway function stimulating agent and whichhave not been contacted with said test compound, thereby selecting acandidate compound; h) contacting cells of neural origin with saidcandidate compound selected in step g) and with a suboptimal dose of aCREB function stimulating agent; i) assessing endogenous CREB-dependentgene expression in the cells which have been contacted with saidcandidate compound and with said CREB function stimulating agent; j)comparing endogenous CREB-dependent gene expression assessed in step i)with endogenous CREB-dependent gene expression in control cells whichhave been contacted with said CREB function stimulating agent and whichhave not been contacted with said candidate compound; k) selecting saidcandidate compound if: 1) endogenous CREB-dependent gene expressionassessed in step i) is increased relative to endogenous CREB-dependentgene expression in control cells which have been contacted with saidCREB function stimulating agent and which have not been contacted withsaid candidate compound; and 2) endogenous CREB-dependent geneexpression in control cells which have not been contacted with said CREBfunction stimulating agent and which have been contacted with saidcandidate compound is not significantly different relative to theCREB-dependent gene expression in control cells which have not beencontacted with said CREB function stimulating agent and which have notbeen contacted with said candidate compound, thereby selecting aconfirmed candidate compound; l) administering said confirmed candidatecompound selected in step k) to an animal; m) training said animaladministered said confirmed candidate compound under conditionsappropriate to produce long term memory formation in said animal; n)assessing long term memory formation in said animal trained in step m);and o) comparing long term memory formation assessed in step n) withlong term memory formation produced in the control animal to which saidconfirmed candidate compound has not been administered.
 20. The methodof claim 19 wherein said host cells are human neuroblastoma cells andsaid cells of neural origin are neurons.
 21. The method of claim 20wherein said neurons are primary hippocampal cells.
 22. The method ofclaim 19 wherein said indicator gene encodes luciferase.
 23. The methodof claim 19 wherein said CREB function stimulating agent is forskolin.24. The method of claim 23 wherein steps a) to e) are repeated with arange of four different concentrations of said test compound selected instep e).
 25. The method of claim 19 wherein said animal is a mammal.